Hematopoietic stem cells (HSC) are defined as clonogenic cells that can give rise to all blood cell lineages, as well as self-renew, at least for a significant period of time. We have identified four subsets of HSCs and multipotent progenitors (MPP), and only the long term HSC (LT-HSC) self renew for the life of the host, while short term (ST)-HSC renew for about 8 weeks and the MPP subsets much less or not at all. We have also prospectively identified by phenotype and isolated two classes of oligopotent progenitors downstream of the MPP - the common lymphoid progenitor (CLP) and the common myeloid progenitor (CMP), and its downstream, further differentiated progeny the GMP, MEP and MKP. In the first section of this grant, dividing LT-HSC, ST-HSC, and MPP will be tested for self-renewal or differentiation to one of the downstream fates at the single cell level to determine if the divisions are symmetrical or asymmetrical, and if the subsequent intermediate stages of differentiation are obligate or optional; i.e., must a LT-HSC go through a ST-HSC stage prior to being an MPP, or is it possible to bypass the presumed hierarchy and differentiate directly into a CMP, for example. Important insights into how the developing HSC makes these decisions will also be gained from studying the molecules and pathways involved in the self-renewal/ differentiation as well as working to identify the niche in the bone marrow where the HSC reside. We have already identified several molecules involved in HSC self-renewal including the wnts, catenins, cadherins, Bmi-1 and Junb. We will use lineage tracing transgenes, lentiviral transduction and RNA interference technologies to manipulate and explore the roles that these and other molecules play in HSC self-renewal and development. In addition, we will use lineage tracing experiments to investigate the cellular intermediates between blastocyst ICMs and HSC in the developing embryo as well as in vitro as derived from embryonic stem cells. This concurrent examination of the purification, development and biology of HSC along with their preclinical utility is the theme that drives the basic research of this grant.